Pathogenesis and Transmission Assessment of 3 Swine-Origin Influenza A(H3N2) Viruses With Zoonotic Risk to Humans Isolated in the United States, 2017-2020.

The sporadic occurrence of human infections with swine-origin influenza A(H3N2) viruses and the continual emergence of novel A(H3N2) viruses in swine herds underscore the necessity for ongoing assessment of the pandemic risk posed by these viruses. Here, we selected 3 recent novel swine-origin A(H3N2) viruses isolated between 2017 to 2020, bearing hemagglutinins from the 1990.1, 2010.1, or 2010.2 clades, and evaluated their ability to cause disease and transmit in a ferret model. We conclude that despite considerable genetic variances, all 3 contemporary swine-origin A(H3N2) viruses displayed a capacity for robust replication in the ferret respiratory tract and were also capable of limited airborne transmission. These findings highlight the continued public health risk of swine-origin A(H3N2) strains, especially in human populations with low cross-reactive immunity.

Reported Global Avian Influenza Detections Among Humans and Animals During 2013-2022: Comprehensive Review and Analysis of Available Surveillance Data.

BACKGROUND: Avian influenza (AI) virus detections occurred frequently in 2022 and continue to pose a health, economic, and food security risk. The most recent global analysis of official reports of animal outbreaks and human infections with all reportable AI viruses was published almost a decade ago. Increased or renewed reports of AI viruses, especially high pathogenicity H5N8 and H5N1 in birds and H5N1, H5N8, and H5N6 in humans globally, have established the need for a comprehensive review of current global AI virus surveillance data to assess the pandemic risk of AI viruses. OBJECTIVE: This study aims to provide an analysis of global AI animal outbreak and human case surveillance information from the last decade by describing the circulating virus subtypes, regions and temporal trends in reporting, and country characteristics associated with AI virus outbreak reporting in animals; surveillance and reporting gaps for animals and humans are identified. METHODS: We analyzed AI virus infection reports among animals and humans submitted to animal and public health authorities from January 2013 to June 2022 and compared them with reports from January 2005 to December 2012. A multivariable regression analysis was used to evaluate associations between variables of interest and reported AI virus animal outbreaks. RESULTS: From 2013 to 2022, 52.2% (95/182) of World Organisation for Animal Health (WOAH) Member Countries identified 34 AI virus subtypes during 21,249 outbreaks. The most frequently reported subtypes were high pathogenicity AI H5N1 (10,079/21,249, 47.43%) and H5N8 (6722/21,249, 31.63%). A total of 10 high pathogenicity AI and 6 low pathogenicity AI virus subtypes were reported to the WOAH for the first time during 2013-2022. AI outbreaks in animals occurred in 26 more Member Countries than reported in the previous 8 years. Decreasing World Bank income classification was significantly associated with decreases in reported AI outbreaks (P<.001-.02). Between January 2013 and June 2022, 17/194 (8.8%) World Health Organization (WHO) Member States reported 2000 human AI virus infections of 10 virus subtypes. H7N9 (1568/2000, 78.40%) and H5N1 (254/2000, 12.70%) viruses accounted for the most human infections. As many as 8 of these 17 Member States did not report a human case prior to 2013. Of 1953 human cases with available information, 74.81% (n=1461) had a known animal exposure before onset of illness. The median time from illness onset to the notification posted on the WHO event information site was 15 days (IQR 9-30 days; mean 24 days). Seasonality patterns of animal outbreaks and human infections with AI viruses were very similar, occurred year-round, and peaked during November through May. CONCLUSIONS: Our analysis suggests that AI outbreaks are more frequently reported and geographically widespread than in the past. Global surveillance gaps include inconsistent reporting from all regions and human infection reporting delays. Continued monitoring for AI virus outbreaks in animals and human infections with AI viruses is crucial for pandemic preparedness.

Interpretation of molecular detection of avian influenza A virus in respiratory specimens collected from live bird market workers in Dhaka, Bangladesh: infection or contamination?

OBJECTIVES: Interpreting real-time reverse transcription-polymerase chain reaction (rRT-PCR) results for human avian influenza A virus (AIV) detection in contaminated settings like live bird markets (LBMs) without serology or viral culture poses a challenge. METHODS: During February-March 2012 and November 2012-February 2013, we screened workers at nine LBMs in Dhaka, Bangladesh, to confirm molecular detections of AIV RNA in respiratory specimens with serology. We tested nasopharyngeal (NP) and throat swabs from workers with influenza-like illness (ILI) and NP, throat, and arm swabs from asymptomatic workers for influenza virus by rRT-PCR and sera for seroconversion and antibodies against HPAI A(H5N1) and A(H9N2) viruses. RESULTS: Among 1273 ILI cases, 34 (2.6%) had A(H5), 56 (4%) had A(H9), and six (0.4%) had both A(H5) and A(H9) detected by rRT-PCR. Of 192 asymptomatic workers, A(H5) was detected in eight (4%) NP and 38 (20%) arm swabs. Of 28 ILI cases with A(H5) or A(H9) detected, none had evidence of seroconversion, but one (3.5%) and 12 (43%) were seropositive for A(H5) and A(H9), respectively. CONCLUSION: Detection of AIV RNA in respiratory specimens from symptomatic and asymptomatic LBM workers without evidence of seroconversion or virus isolation suggests environmental contamination, emphasizing caution in interpreting rRT-PCR results in high viral load settings.

Swine-to-Ferret Transmission of Antigenically Drifted Contemporary Swine H3N2 Influenza A Virus Is an Indicator of Zoonotic Risk to Humans.

Human-to-swine transmission of influenza A (H3N2) virus occurs repeatedly and plays a critical role in swine influenza A virus (IAV) evolution and diversity. Human seasonal H3 IAVs were introduced from human-to-swine in the 1990s in the United States and classified as 1990.1 and 1990.4 lineages; the 1990.4 lineage diversified into 1990.4.A-F clades. Additional introductions occurred in the 2010s, establishing the 2010.1 and 2010.2 lineages. Human zoonotic cases with swine IAV, known as variant viruses, have occurred from the 1990.4 and 2010.1 lineages, highlighting a public health concern. If a variant virus is antigenically drifted from current human seasonal vaccine (HuVac) strains, it may be chosen as a candidate virus vaccine (CVV) for pandemic preparedness purposes. We assessed the zoonotic risk of US swine H3N2 strains by performing phylogenetic analyses of recent swine H3 strains to identify the major contemporary circulating genetic clades. Representatives were tested in hemagglutination inhibition assays with ferret post-infection antisera raised against existing CVVs or HuVac viruses. The 1990.1, 1990.4.A, and 1990.4.B.2 clade viruses displayed significant loss in cross-reactivity to CVV and HuVac antisera, and interspecies transmission potential was subsequently investigated in a pig-to-ferret transmission study. Strains from the three lineages were transmitted from pigs to ferrets via respiratory droplets, but there were differential shedding profiles. These data suggest that existing CVVs may offer limited protection against swine H3N2 infection, and that contemporary 1990.4.A viruses represent a specific concern given their widespread circulation among swine in the United States and association with multiple zoonotic cases.

Cross-neutralization and viral fitness of SARS-CoV-2 Omicron sublineages.

The rapid evolution of SARS-CoV-2 Omicron sublineages mandates a better understanding of viral replication and cross-neutralization among these sublineages. Here we used K18-hACE2 mice and primary human airway cultures to examine the viral fitness and antigenic relationship among Omicron sublineages. In both K18-hACE2 mice and human airway cultures, Omicron sublineages exhibited a replication order of BA.5 ≥ BA.2 ≥ BA.2.12.1 > BA.1; no difference in body weight loss was observed among different sublineage-infected mice. The BA.1-, BA.2-, BA.2.12.1-, and BA.5-infected mice developed distinguishable cross-neutralizations against Omicron sublineages, but exhibited little neutralization against the index virus (i.e. USA-WA1/2020) or the Delta variant. Surprisingly, the BA.5-infected mice developed higher neutralization activity against heterologous BA.2 and BA.2.12.1 than that against homologous BA.5; serum neutralizing titres did not always correlate with viral replication levels in infected animals. Our results revealed a distinct antigenic cartography of Omicron sublineages and support the bivalent vaccine approach.

Influenza A(H7N9) Pandemic Preparedness: Assessment of the Breadth of Heterologous Antibody Responses to Emerging Viruses from Multiple Pre-Pandemic Vaccines and Population Immunity.

Influenza A(H7N9) viruses remain as a high pandemic threat. The continued evolution of the A(H7N9) viruses poses major challenges in pandemic preparedness strategies through vaccination. We assessed the breadth of the heterologous neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses using the 1st wave vaccine sera from 4 vaccine groups: 1. inactivated vaccine with 2.8 µg hemagglutinin (HA)/dose + AS03A; 2. inactivated vaccine with 5.75 µg HA/dose + AS03A; 3. inactivated vaccine with 11.5 µg HA/dose + MF59; and 4. recombinant virus like particle (VLP) vaccine with 15 µg HA/dose + ISCOMATRIX™. Vaccine group 1 had the highest antibody responses to the vaccine virus and the 3rd/5th wave drifted viruses. Notably, the relative levels of cross-reactivity to the drifted viruses as measured by the antibody GMT ratios to the 5th wave viruses were similar across all 4 vaccine groups. The 1st wave vaccines induced robust responses to the 3rd and Pearl River Delta lineage 5th wave viruses but lower cross-reactivity to the highly pathogenic 5th wave A(H7N9) virus. The population in the United States was largely immunologically naive to the A(H7N9) HA. Seasonal vaccination induced cross-reactive neuraminidase inhibition and binding antibodies to N9, but minimal cross-reactive antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies to A(H7N9).

Interspecies Transmission from Pigs to Ferrets of Antigenically Distinct Swine H1 Influenza A Viruses with Reduced Reactivity to Candidate Vaccine Virus Antisera as Measures of Relative Zoonotic Risk.

During the last decade, endemic swine H1 influenza A viruses (IAV) from six different genetic clades of the hemagglutinin gene caused zoonotic infections in humans. The majority of zoonotic events with swine IAV were restricted to a single case with no subsequent transmission. However, repeated introduction of human-seasonal H1N1, continual reassortment between endemic swine IAV, and subsequent drift in the swine host resulted in highly diverse swine IAV with human-origin genes that may become a risk to the human population. To prepare for the potential of a future swine-origin IAV pandemic in humans, public health laboratories selected candidate vaccine viruses (CVV) for use as vaccine seed strains. To assess the pandemic risk of contemporary US swine H1N1 or H1N2 strains, we quantified the genetic diversity of swine H1 HA genes, and identified representative strains from each circulating clade. We then characterized the representative swine IAV against human seasonal vaccine and CVV strains using ferret antisera in hemagglutination inhibition assays (HI). HI assays revealed that 1A.3.3.2 (pdm09) and 1B.2.1 (delta-2) demonstrated strong cross reactivity to human seasonal vaccines or CVVs. However, swine IAV from three clades that represent more than 50% of the detected swine IAVs in the USA showed significant reduction in cross-reactivity compared to the closest CVV virus: 1A.1.1.3 (alpha-deletion), 1A.3.3.3-clade 3 (gamma), and 1B.2.2.1 (delta-1a). Representative viruses from these three clades were further characterized in a pig-to-ferret transmission model and shown to exhibit variable transmission efficiency. Our data prioritize specific genotypes of swine H1N1 and H1N2 to further investigate in the risk they pose to the human population.

Differential neutralization and inhibition of SARS-CoV-2 variants by antibodies elicited by COVID-19 mRNA vaccines.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccine-mediated protection from disease. To ascertain and rank the risk of VOCs and VOIs, we analyze the ability of 14 variants (614G, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Kappa, Lambda, Mu, and Omicron) to escape from mRNA vaccine-induced antibodies. The variants show differential reductions in neutralization and replication by post-vaccination sera. Although the Omicron variant (BA.1, BA.1.1, and BA.2) shows the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retain moderate neutralizing activity against that variant. Therefore, vaccination remains an effective strategy during the COVID-19 pandemic.

Pathogenesis and transmission of human seasonal and swine-origin A(H1) influenza viruses in the ferret model.

Influenza A viruses (IAVs) in the swine reservoir constantly evolve, resulting in expanding genetic and antigenic diversity of strains that occasionally cause infections in humans and pose a threat of emerging as a strain capable of human-to-human transmission. For these reasons, there is an ongoing need for surveillance and characterization of newly emerging strains to aid pandemic preparedness efforts, particularly for the selection of candidate vaccine viruses and conducting risk assessments. Here, we performed a parallel comparison of the pathogenesis and transmission of genetically and antigenically diverse swine-origin A(H1N1) variant (v) and A(H1N2)v, and human seasonal A(H1N1)pdm09 IAVs using the ferret model. Both groups of viruses were capable of replication in the ferret upper respiratory tract; however, variant viruses were more frequently isolated from the lower respiratory tract as compared to the human-adapted viruses. Regardless of virus origin, observed clinical signs of infection differed greatly between strains, with some viruses causing nasal discharge, sneezing and, in some instances, diarrhea in ferrets. The most striking difference between the viruses was the ability to transmit through the air. Human-adapted viruses were capable of airborne transmission between all ferret pairs. In contrast, only one out of the four tested variant viruses was able to transmit via the air as efficiently as the human-adapted viruses. Overall, this work highlights the need for sustained monitoring of emerging swine IAVs to identify strains of concern such as those that are antigenically different from vaccine strains and that possess adaptations required for efficient respiratory droplet transmission in mammals.

Influenza A virus circulation in backyard animals in the Pacific coast of Guatemala, 2013-2014.

Due to their documented epidemiological relevance as hosts for influenza A viruses (IAV), humans, poultry and pigs in backyard production systems (BPS) within wetlands could be key to the emergence of novel IAV variants able to transmit between humans or animals. To better understand the circulation of IAV at the human-animal interface of BPS within wetlands, we studied IAV in backyard duck flocks and pig herds in the Pacific Coast of Guatemala. From April 2013 to October 2014, we estimated the monthly IAV per cent seropositive and viral positive flocks and herds in two resource-limited communities. We detected antibodies in sera against the IAV nucleoprotein through ELISA. We also detected IAV viral RNA in respiratory (ducks and pigs) and cloacal (ducks) swabs through rRT-PCR directed at the matrix gene. We attempted viral isolation in eggs or MDCK cells followed by sequencing from swabs positive for IAV. During our study period, IAV seropositivity in duck flocks was 38%, and viral positivity was 23% (n = 86 BPS sampled). IAV seropositivity in pig herds was 42%, and viral positivity was 20% (n = 90 BPS sampled). Both flocks and herds had detectable antibodies against IAV mostly year-round, and IAV was detected in several months. We isolated an H3N2 virus from one pig sampled at the end of 2013. Standard nucleotide BLAST searches indicate that the isolated virus was similar to seasonal viruses circulating in humans, suggesting human-to-pig transmission. Our data show concurrent circulation of IAV in multiple species of poultry and pigs that were commingled in rudimentary conditions in proximity to humans, but no significant risk factors could be identified.

Crossroads of highly pathogenic H5N1: overlap between wild and domestic birds in the Black Sea-Mediterranean impacts global transmission.

Understanding transmission dynamics that link wild and domestic animals is a key element of predicting the emergence of infectious disease, an event that has highest likelihood of occurring wherever human livelihoods depend on agriculture and animal trade. Contact between poultry and wild birds is a key driver of the emergence of highly pathogenic avian influenza (HPAI), a process that allows for host switching and accelerated reassortment, diversification, and spread of virus between otherwise unconnected regions. This study addresses questions relevant to the spillover of HPAI at a transmission hotspot: what is the nature of the wild bird-poultry interface in Egypt and adjacent Black Sea-Mediterranean countries and how has this contributed to outbreaks occurring worldwide? Using a spatiotemporal model of infection risk informed by satellite tracking of waterfowl and viral phylogenetics, this study identified ecological conditions that contribute to spillover in this understudied region. Results indicated that multiple ducks (Northern Shoveler and Northern Pintail) hosted segments that shared ancestry with HPAI H5 from both clade 2.2.1 and clade 2.3.4 supporting the role of Anseriformes in linking viral populations in East Asia and Africa over large distances. Quantifying the overlap between wild ducks and H5N1-infected poultry revealed an increasing interface in late winter peaking in early spring when ducks expanded their range before migration, with key differences in the timing of poultry contact risk between local and long-distance migrants.

Changes in Influenza and Other Respiratory Virus Activity During the COVID-19 Pandemic - United States, 2020-2021.

The COVID-19 pandemic and subsequent implementation of nonpharmaceutical interventions (e.g., cessation of global travel, mask use, physical distancing, and staying home) reduced transmission of some viral respiratory pathogens (1). In the United States, influenza activity decreased in March 2020, was historically low through the summer of 2020 (2), and remained low during October 2020-May 2021 (<0.4% of respiratory specimens with positive test results for each week of the season). Circulation of other respiratory pathogens, including respiratory syncytial virus (RSV), common human coronaviruses (HCoVs) types OC43, NL63, 229E, and HKU1, and parainfluenza viruses (PIVs) types 1-4 also decreased in early 2020 and did not increase until spring 2021. Human metapneumovirus (HMPV) circulation decreased in March 2020 and remained low through May 2021. Respiratory adenovirus (RAdV) circulated at lower levels throughout 2020 and as of early May 2021. Rhinovirus and enterovirus (RV/EV) circulation decreased in March 2020, remained low until May 2020, and then increased to near prepandemic seasonal levels. Circulation of respiratory viruses could resume at prepandemic levels after COVID-19 mitigation practices become less stringent. Clinicians should be aware of increases in some respiratory virus activity and remain vigilant for off-season increases. In addition to the use of everyday preventive actions, fall influenza vaccination campaigns are an important component of prevention as COVID-19 mitigation measures are relaxed and schools and workplaces resume in-person activities.

Swine Influenza A Viruses and the Tangled Relationship with Humans.

Influenza A viruses (IAVs) are the causative agents of one of the most important viral respiratory diseases in pigs and humans. Human and swine IAV are prone to interspecies transmission, leading to regular incursions from human to pig and vice versa. This bidirectional transmission of IAV has heavily influenced the evolutionary history of IAV in both species. Transmission of distinct human seasonal lineages to pigs, followed by sustained within-host transmission and rapid adaptation and evolution, represent a considerable challenge for pig health and production. Consequently, although only subtypes of H1N1, H1N2, and H3N2 are endemic in swine around the world, extensive diversity can be found in the hemagglutinin (HA) and neuraminidase (NA) genes, as well as the remaining six genes. We review the complicated global epidemiology of IAV in swine and the inextricably entangled implications for public health and influenza pandemic planning.

Laboratory evaluation of two point-of-care detection systems for early and accurate detection of influenza viruses in the Lao People's Democratic Republic.

BACKGROUND: We evaluated molecular-based point-of-care influenza virus detection systems in a laboratory prior to a field evaluation of on-site specimen testing. METHODS: The performance characteristics of 1) insulated isothermal polymerase chain reaction (PCR) on a POCKIT™ device and 2) real-time reverse transcription-PCR (rRT-PCR) on a MyGo Mini™ device were evaluated using human clinical specimens, beta-propiolactone-inactivated influenza viruses, and RNA controls. The rRT-PCR carried out on a CXF-96™ real-time detection system was used as a gold standard for comparison. RESULTS: Both systems demonstrated 100% sensitivity and specificity and test results were in 100% agreement with the gold standard. POCKIT™ only correctly identified influenza A (M gene) in clinical specimens due to the unavailability of typing and subtyping reagents for human influenza viruses, while MyGo Mini™ had either a one log higher or the same sensitivity in detecting influenza viruses in clinical specimens compared to the gold standard. For inactivated viruses and/or viral RNA, the analytic sensitivity of POCKIT™ was shown to be comparable to, or more sensitive, than the gold standard. The analytic sensitivity of MyGo Mini™ had mixed results depending on the types and subtypes of influenza viruses. CONCLUSIONS: The performance of the two systems in a laboratory is promising and supports further evaluation in field settings.

Detection and Characterization of Swine Origin Influenza A(H1N1) Pandemic 2009 Viruses in Humans following Zoonotic Transmission.

Human-to-swine transmission of seasonal influenza viruses has led to sustained human-like influenza viruses circulating in the U.S. swine population. While some reverse zoonotic-origin viruses adapt and become enzootic in swine, nascent reverse zoonoses may result in virus detections that are difficult to classify as "swine-origin" or "human-origin" due to the genetic similarity of circulating viruses. This is the case for human-origin influenza A(H1N1) pandemic 2009 (pdm09) viruses detected in pigs following numerous reverse zoonosis events since the 2009 pandemic. We report the identification of two human infections with A(H1N1)pdm09 viruses originating from swine hosts and classify them as "swine-origin" variant influenza viruses based on phylogenetic analysis and sequence comparison methods. Phylogenetic analyses of viral genomes from two cases revealed these viruses were reassortants containing A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) genes with genetic combinations derived from the triple reassortant internal gene cassette. Follow-up investigations determined that one individual had direct exposure to swine in the week preceding illness onset, while another did not report swine exposure. The swine-origin A(H1N1) variant cases were resolved by full genome sequence comparison of the variant viruses to swine influenza genomes. However, if reassortment does not result in the acquisition of swine-associated genes and swine virus genomic sequences are not available from the exposure source, future cases may not be discernible. We have developed a pipeline that performs maximum likelihood analyses, a k-mer-based set difference algorithm, and random forest algorithms to identify swine-associated sequences in the hemagglutinin gene to differentiate between human-origin and swine-origin A(H1N1)pdm09 viruses.IMPORTANCE Influenza virus infects a wide range of hosts, resulting in illnesses that vary from asymptomatic cases to severe pneumonia and death. Viral transfer can occur between human and nonhuman hosts, resulting in human and nonhuman origin viruses circulating in novel hosts. In this work, we have identified the first case of a swine-origin influenza A(H1N1)pdm09 virus resulting in a human infection. This shows that these viruses not only circulate in swine hosts, but are continuing to evolve and distinguish themselves from previously circulating human-origin influenza viruses. The development of techniques for distinguishing human-origin and swine-origin viruses are necessary for the continued surveillance of influenza viruses. We show that unique genetic signatures can differentiate circulating swine-associated strains from circulating human-associated strains of influenza A(H1N1)pdm09, and these signatures can be used to enhance surveillance of swine-origin influenza.

Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models.

Low-pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Cocirculation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses.IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.

Mammalian pathogenicity and transmissibility of low pathogenic avian influenza H7N1 and H7N3 viruses isolated from North America in 2018.

ABSTRACTLow pathogenic avian influenza (LPAI) H7 subtype viruses are infrequently, but persistently, associated with outbreaks in poultry in North America. These LPAI outbreaks provide opportunities for the virus to develop enhanced virulence and transmissibility in mammals and have previously resulted in both occasional acquisition of a highly pathogenic avian influenza (HPAI) phenotype in birds and sporadic cases of human infection. Two notable LPAI H7 subtype viruses caused outbreaks in 2018 in North America: LPAI H7N1 virus in chickens and turkeys, representing the first confirmed H7N1 infection in poultry farms in the United States, and LPAI H7N3 virus in turkeys, a virus subtype often associated with LPAI-to-HPAI phenotypes. Here, we investigated the replication capacity of representative viruses from these outbreaks in human respiratory tract cells and mammalian pathogenicity and transmissibility in the mouse and ferret models. We found that the LPAI H7 viruses replicated to high titre in human cells, reaching mean peak titres generally comparable to HPAI H7 viruses. Replication was efficient in both mammalian species, causing mild infection, with virus primarily limited to respiratory tract tissues. The H7 viruses demonstrated a capacity to transmit to naïve ferrets in a direct contact setting. These data support the need to perform routine risk assessments of LPAI H7 subtype viruses, even in the absence of confirmed human infection.

Development of an RNA Strand-Specific Hybridization Assay To Differentiate Replicating versus Nonreplicating Influenza A Viruses.

Replication of influenza A virus (IAV) from negative-sense viral RNA (vRNA) requires the generation of positive-sense RNA (+RNA). Most molecular assays, such as conventional real-time reverse transcriptase PCR (rRT-PCR), detect total RNA in a sample without differentiating vRNA from +RNA. These assays are not designed to distinguish IAV infection versus exposure of an individual to an environment enriched with IAVs but wherein no viral replication occurs. We therefore developed a strand-specific hybridization (SSH) assay that differentiates between vRNA and +RNA and quantifies relative levels of each RNA species. The SSH assay exhibited a linearity of 7 logs with a lower limit of detection of 6.0 × 102 copies of molecules per reaction. No signal was detected in samples with a high load of nontarget template or influenza B virus, demonstrating assay specificity. IAV +RNA was detected 2 to 4 h postinoculation of MDCK cells, whereas synthesis of cold-adapted IAV +RNA was significantly impaired at 37°C. The SSH assay was then used to test IAV rRT-PCR positive nasopharyngeal specimens collected from individuals exposed to IAV at swine exhibitions (n = 7) or while working at live bird markets (n = 2). The SSH assay was able to differentiate vRNA and +RNA in samples collected from infected, symptomatic individuals versus individuals who were exposed to IAV in the environment but had no active viral replication. Data generated with this technique, especially when coupled with clinical data and assessment of seroconversion, will facilitate differentiation of actual IAV infection with replicating virus versus individuals exposed to high levels of environmental contamination but without virus infection.